The Hedgehog (Hh) signaling pathway is vital for proper patterning and organ development during embryogenesis. Aberrant Hh signaling activity causes numerous developmental diseases and plays a pivotal role in the pathogenesis of several cancers, including basal cell carcinoma, rhabdomyosarcoma, and medulloblastoma. More recent studies implicate Hh signaling in multiple additional cancers, including pancreatic cancer, where tumor cell derived Hh ligands signal in a paracrine manner to mediate essential interactions with the surrounding microenvironment. Strikingly, pancreatic cancer is characterized by an extensive Hh-driven fibrotic stroma, believed to be responsible for the chemo-resistance of this tumor. However, remarkably little is known about the extracellular signaling components that drive oncogenic Hh signaling. We propose to fill this major gap in our knowledge through a systematic analysis of three novel, cell surface molecules that are required for Hh signaling: Gas1, Cdo and Boc. Recent studies from the Allen lab indicate that Gas1, Cdo and Boc represent novel Hh co-receptors that are essential for Hh pathway activation in multiple tissues during embryogenesis. Work from the Pasca di Magliano lab has identified an important role for Hh signaling in pancreatic cancer. Thus, a central question is: what roles do these novel receptors play in the pathologic activation of Hh signaling in cancer? The long-term goal of this research is to develop new therapeutic approaches to the treatment of pancreatic cancer. This proposal is a unique collaboration between the Allen and Pasca di Magliano labs that combines expertise in Hh signaling and pancreatic cancer to test the hypothesis that that Gas1, Cdo and Boc are essential regulators of Hh ligand-driven pancreatic cancer. The objectives of this proposal are to: 1) determine the expression of Gas1, Cdo and Boc in normal and diseased pancreas, 2) interrogate their function in pancreatic cancer and 3) generate new tools to modulate the Hh pathway by targeting these novel cell surface receptors. Aim 1 will determine the expression of Gas1, Cdo and Boc in adult pancreas and pancreatic cancer and functionally assess their function as stromal regulators of pancreatic cancer. Aim 2 will develop and functionally validate novel inhibitors of Hh receptors in pancreatic cancer. The expected outcomes of the proposed work include: 1) a detailed analysis of Gas1, Cdo and Boc expression in the adult pancreas and pancreatic cancer, 2) a comprehensive assessment of Gas1, Cdo and Boc function as stromal regulators of Hh signaling in pancreatic cancer, and 3) the generation of a new set of therapeutic tools to selectively modulate Hh pathway activity in pancreatic cancer. We expect that these results will have a significant positive impact on determining the fundamental roles of Gas1, Cdo and Boc in pancreatic cancer and on the development of compounds targeting these novel Hh receptors as an alternative approach to treat pancreatic cancer, which currently lacks effective treatments, as well as other Hh ligand-driven cancers.